Adiponectin is a hormone which is produced and secreted specifically in fatty tissue and having anti-diabetes and anti-arteriosclerosis activity, and is present in blood at a relatively high level. In recent years, hypoadiponectinemia associated with obesity, particularly caused by accumulation of visceral fat, is thought to trigger onset of diabetes, arteriosclerotic diseases or hypertension.
Structurally, adiponectin belongs to the Clq (Complement lq) family and has a collagen-like domain which is intrinsic to the Clq family. A study has reported that adiponectin forms a multimer mainly composed of trimers. Recently, the present inventors have revealed the structure of adiponectin multimers present in human blood (trimer including albumin-bound trimer, hexamer, and HMW fraction), and have reported that, among adiponectin multimers, HMW adiponectin exhibits the highest activation represented by phosphorylation activity on AMPK (adenosine monophosphate activated protein kinase) which promotes glucose intake and fatty acid metabolism (Non-Patent Document 1). In addition, the inventors disclosed a method of directly determining the HMW adiponectin level and indirectly determining the level of a trimer fraction or a hexamer fraction, respectively, in which a biological sample containing human-derived adiponectin multimers is reacted with a specific type of protease to selectively digest fractions other than the target fraction and remaining adiponectins are immunologically assayed (Patent Document 1 and Non-Patent Document 2).
A clinical study on adiponectin multimers in diabetes groups and coronary artery disease groups through employment of the selective assay system has revealed that the ratio of HMW adiponectin level to total adiponectin level (HMWR) is a more sensitive and specific prophetic index for insulin resistance and metabolic syndrome than is the total adiponectin level (Non-Patent Document 3). Thus, not only an assay of the total adiponectin level but also a selective assay of fractions is envisaged to be clinically useful.
Meanwhile, among therapeutic drugs for metabolic-syndrome-related diseases (diabetes, hypertension, and hyperlipidemea), some drugs increase adiponectin, and are of interest. For example, a thiazolidine derivative, which is an insulin resistance ameliorating agent, increases HMW adiponectin (Non-Patent Document 4). In addition, recently, a drug categorized as an angiotensin receptor antagonist which is used as a hypotensive drug and some therapeutic agents for hyperlipidemea are reported to increase adiponectin (Non-Patent Document 5). Thus, adiponectin is expected to ameliorate insulin-resistance which is frequently observed in disease groups involving metabolic syndrome.
As described above, adiponectin-increasing action is of great value in the development of drugs for metabolic-syndrome-related diseases. In addition to the drug development, adiponectin attracts attention in the field of healthy food, and research and development of functional food having adiponectin-increasing action is extensively carried out.
Generally, the efficacy and effect of a drug or a functional food is assessed primarily through experiments by use of experimental animals such as mice and rats. Regarding adiponectin-increasing action, the change in the blood adiponectin level of the experimental animal is effectively employed as an index (see, for example, Patent Documents 2 and 3). The adiponectin level in samples of a mouse or a rat is available by means of a commercially available assay kit. However, currently available kits can only determine the total adiponectin level but cannot selectively determine levels of adiponectin multimers. Particularly, there is demand for a fractional assay method for highly active HMW adiponectin.
The present inventors found a method of selectively assaying adiponectin multimers in which adiponectin multimers present in a human biological sample are reacted with a specific type of protease (Patent Document 1 and Non-Patent Document 2). Interestingly, the inventors found that when the protease used in the method was applied in an assay of mouse-derived adiponectin multimers, precise results were not obtained particularly for assaying an HMW fraction.
Pajvani et al. reported digestion specificity of trypsin on recombinant mouse-derived adiponectins. According to this report, trypsin digests a low-molecular-weight fraction (LMW) and does not digest a middle-molecular-weight fraction (MMW) or an HMW fraction. Thus, trypsin is not suited for selective measurement of HMW adiponectin (Non-Patent Document 6).